CN103480340A - Immunoaffinity absorbent for enriching ochratoxin A, preparation method and application thereof - Google Patents
Immunoaffinity absorbent for enriching ochratoxin A, preparation method and application thereof Download PDFInfo
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Abstract
The present invention discloses an immunoaffinity absorbent for enriching ochratoxin A, a preparation method and an application thereof. The immunoaffinity absorbent is a conjugate obtained through conjugating ochratoxin A polyclonal antibody and an amino-containing solid phase carrier, wherein the ochratoxin A polyclonal antibody is prepared according to the following steps: adopting an ochratoxin A-NHS active ester and carrier protein conjugate as immunogen to immunize non-human mammal to obtain antiserum, and purifying the antiserum to obtain the ochratoxin A polyclonal antibody. The immunoaffinity absorbent for enriching the ochratoxin A and the affinity chromatography column thereof have the following advantages that: ochratoxin A concentration in a sample requiring detection can be concentrated, a detection lower limitation is increased, impurity interference can be eliminated, detection accuracy and reliability can be increased, and a sample purifying time through the column is reduced so as to achieve rapid detection.
Description
Technical field
The present invention relates to the immune affinity sorbent and preparation method thereof and application of enrichment ochratoxin A.
Background technology
Ochratoxin is that in Aspergillus ochraceus and Penicillium notatum, the toxic metabolite product produced after grain, food, feed and other agricultural byproducts is infected in some toxigenic bacterium strain.Ochratoxin A is a kind of that the ochratoxin Poisoning is the strongest, output is the highest, and humans and animals is had to strong toxic action, can cause nephrarctia, fetal anomaly, miscarriage, the death of animal, and has the carcinogenicity of height.Ochratoxin A exists in most of cereal such as barley, wheat, oat, corn.The poultry of raising with the cereal that polluted by ochratoxin A also can be polluted, and ochratoxin A directly or indirectly jeopardizes health.Therefore, ochratoxin is another toxin that causes World Focusing after aflatoxin.1993, international cancer research institute (The International Agency for Researchon Cancer) was decided to be 2B group carcinogen (may be mankind's carcinogenic substance) by ochratoxin.Various countries have formulated the limit standard of ochratoxin A in cereal one after another, the 56th FAO/WHO food additives joint specialist executive session carried out hazard assessment to ochratoxin A and formulated ochratoxin A maximum residue limit standard in cereal foods is 5 μ g/kg, and it is 5 μ g/kg that China and European Union and member state thereof also are defined in maximum residue limit in cereal.
The assay method of current ochratoxin A has multiple, as: TLC (TLC), high performance liquid chromatography (HPLC) and the methods such as LC/MS (LC/MS), immunology detection.Due to the substance in biological sample complexity, testing concentration is low, and most of sampling amounts are few.This just has higher requirement to sensitivity, the degree of accuracy of analytical method.The immunoaffinity chromatography technology is a kind of analytical method that immune response and chromatographic technique combine that collects, efficiency is high, purity is high, technology fast on purifying substances to be one, can make immunoassay technology (as ELISA etc.) and chromatographic technique obtain complementation aspect specificity, separating power, speed and sensitivity, avoid the deficiency of the direct working sample of immunoassay.Use the ochratoxin A in immune affinity chromatographic purification enrichment sample, can improve significantly the limit of precision of analysis, reliability and analysis.
Summary of the invention
Technical problem to be solved by this invention overcomes the deficiencies in the prior art, and the immune affinity sorbent and preparation method thereof of the ochratoxin A of a kind of high column capacity (high accumulation rate) is provided.
The immune affinity sorbent of enrichment ochratoxin A provided by the present invention is by the ochratoxin A polyclonal antibody and contain the conjugate (adsorbent) that amino solid phase carrier coupling obtains;
Described ochratoxin A polyclonal antibody can be according to the preparation of the method that comprises the steps: the conjugate of ochratoxin A-NHS active ester and carrier protein of take obtains antiserum as the immunogen immune non-human mammal, and from described antiserum, purifying obtains described enrichment ochratoxin A polyclonal antibody.
Wherein, described non-human mammal can be rabbit, sheep, mouse, cavy or horse etc.
In the immune affinity sorbent of above-mentioned enrichment ochratoxin A, described ochratoxin A-NHS active ester is that ochratoxin A passes through N, N-dicyclohexyl carbodiimide (N, N'-dicyclohexylcarbodiimide, DCC) react with N-hydroxy-succinamide (N-Hydroxysuccinimide, NHS) active ester generated.
In the immune affinity sorbent of above-mentioned enrichment ochratoxin A, can prepare according to the method comprised the steps by described ochratoxin A-NHS active ester:
1) iodoacetic acid is passed through to N, N-dicyclohexyl carbodiimide (N, N'-dicyclohexylcarbodiimide, DCC) and N-hydroxy-succinamide (N-Hydroxysuccinimide, NHS) reaction generates active ester, and described active ester is NHS-iodacetyl active ester;
2) described NHS-iodacetyl active ester and ochratoxin A are carried out to substitution reaction, obtain ochratoxin A-NHS active ester.
In the immune affinity sorbent of above-mentioned enrichment ochratoxin A, described 1) can be iodoacetic acid, N-hydroxy-succinamide and N, the N-dicyclohexyl carbodiimide carries out condensation reaction in the dark, obtains NHS-iodacetyl active ester.Further, described 1) can be iodoacetic acid, N-hydroxy-succinamide and N, the N-dicyclohexyl carbodiimide is with the mol ratio of 269:695:582, in oxolane, 20 ℃-25 ℃ are carried out condensation reaction in the dark, after completion of the reaction, centrifugal with 10000g, get supernatant and obtain NHS-iodacetyl active ester;
Described 2) can be the tetrahydrofuran solution of ochratoxin A and described supernatant (described NHS-iodacetyl active ester) are mixed, at 20-25 ℃, carry out substitution reaction, obtain described ochratoxin A-NHS active ester.
In the immune affinity sorbent of above-mentioned enrichment ochratoxin A, the method that purifying obtains described enrichment ochratoxin A polyclonal antibody from described antiserum comprises: using described ochratoxin A-NHS active ester and the described conjugate obtained containing amino solid phase carrier coupling as the affinity chromatography medium of the described ochratoxin A polyclonal antibody of purifying, from described antiserum, by affinitive layer purification, obtain described ochratoxin A polyclonal antibody; And/or
The described ochratoxin A Anti-TNF-α of the acetic acid aqueous solution wash-out that is 3% by concentration expressed in percentage by volume in described affinity chromatography.
In the immune affinity sorbent of above-mentioned enrichment ochratoxin A, describedly containing amino solid phase carrier, can be containing amino Ago-Gel or for containing amino sephadex; And/or,
Described carrier protein is hemocyanin or bovine serum albumin(BSA).
The method for preparing the immune affinity sorbent of above-mentioned enrichment ochratoxin A also belongs to protection scope of the present invention.
The kit of the immune affinity chromatographic column of the enrichment ochratoxin A that the above-mentioned immune affinity sorbent of take is filler, the enrichment ochratoxin A that contains above-mentioned immune affinity sorbent or above-mentioned immune affinity chromatographic column also belongs to protection scope of the present invention.
Described kit also comprises sample-loading buffer, rinsing liquid and eluent.Described sample-loading buffer can be PBS, and described rinsing liquid can be PBSt, and described eluent can be acetic acid and methanol aqueous solution, the solvent of described acetic acid and methanol aqueous solution is water, solute is acetic acid and methyl alcohol, and the concentration expressed in percentage by volume of acetic acid is 3%, and the concentration expressed in percentage by volume of methyl alcohol is 80%.
Every liter of described PBS is by NaCl 8.0g, KCl 0.2g, KH
2pO
40.2g, Na
2hPO
4.12H
2o 2.9g, add steaming water and be settled to 1000mL, and adjust pH to 7.4 is made.
Every liter of described PBSt is by NaCl 8.0g, KCl 0.2g, KH
2pO
40.2g, Na
2hPO
4.12H
2o 2.9g, Tween-20 500uL, add steaming water and be settled to 1000mL, and adjust pH to 7.4 is made.
Above-mentioned immune affinity sorbent, above-mentioned immune affinity chromatographic column or the application of mentioned reagent box in the enrichment ochratoxin A also belong to protection scope of the present invention.
Described enrichment ochratoxin A specifically can be the ochratoxin A in enrichment cereal or feed.
In above-mentioned application, comprise the step obtained as follows for the sample solution of upper prop:
Cereal or feed are ground and by the testing sieve of 1mm, not pulverize.Take the sample of 5g (being accurate to 0.01g) grinding in the 15mL centrifuge tube, add 1g sodium chloride and 10mL extract (80% methanol aqueous solution), twist good lid, concussion at a high speed, extraction 5min; Then the centrifugal 5min of 4000rpm, accurately pipette the 4mL supernatant in the centrifuge tube of 50mL, adds every liter of PBS of PBS(by NaCl 8.0g, KCl 0.2g, KH
2pO
40.2g, Na
2hPO
4.12H
2o2.9g, add steaming water and be settled to 1000mL, and adjust pH to 7.4 is made) be diluted to scale, mix, through glass fiber filter paper, filter, obtain the sample solution for upper prop.
In above-mentioned application, also comprise the above-mentioned sample solution for upper prop is carried out to the step of enrichment in accordance with the following steps: the immune affinity chromatographic column of the above-mentioned sample solution for upper prop being crossed to above-mentioned enrichment ochratoxin A, then with described rinsing liquid washing, use again described eluent wash-out, obtain the ochratoxin A solution of enrichment.
The immune affinity sorbent that experiment showed, enrichment ochratoxin A of the present invention just has very high accumulation rate, sensitivity and the rate of recovery to ochratoxin A.The immune affinity sorbent that is 52 μ g/mL enrichment ochratoxin As to the accumulation rate of ochratoxin A, can reach 100ng/L to ochratoxin A sensitivity, and the rate of recovery of ochratoxin A is reached more than 80%.The immune affinity sorbent of enrichment ochratoxin A of the present invention can be efficient, accurate, reliable, fast and convenient the enrichment ochratoxin A.The immune affinity sorbent of the enrichment ochratoxin A of the present invention of dress column volume 25 μ L can reach the capacity of existing purification enrichment post dress column volume 1000 μ L and more responsive bioaccumulation efficiency.The immune affinity sorbent of enrichment ochratoxin A of the present invention can get up ochratoxin A enrichment in sample to be checked, in order to further be applied to the quick test that chemical analysis instrument detects (HPLC, high performance liquid chromatography) and colloidal gold test strip.The immune affinity sorbent of enrichment ochratoxin A of the present invention and affinity chromatographic column thereof have ochratoxin A concentration in concentrated testing sample, improve detection lower limit, despumation interference, raising detection accuracy and reliability, minimizing sample and cross the advantage that the post time reaches fast detecting.
The accompanying drawing explanation
The ochratoxin A that Fig. 1 purifies for the immune affinity chromatographic column with enrichment ochratoxin A of the present invention, the design sketch obtained through the colloidal gold strip test.
In figure, A:PBS is that C, T all develop the color through the colloidal gold strip testing result, negative; The ochratoxin A solution of B:0.1ng/mL is that C, T all develop the color through the colloidal gold strip testing result, negative; The ochratoxin A solution of C:0.1ng/mL carries out affinity chromatography collection efflux (filtered solution) as sample is that C, T all develop the color through the colloidal gold strip testing result, negative; It is that C colour developing, T do not develop the color through the colloidal gold strip testing result that the ochratoxin A solution of D:0.1ng/mL carries out as sample the liquid that affinity chromatography elutes, positive.
The specific embodiment
Below in conjunction with the specific embodiment, the present invention is further described in detail, the embodiment provided is only in order to illustrate the present invention, rather than in order to limit the scope of the invention.Experimental technique in following embodiment, if no special instructions, be conventional method.In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
In following embodiment, the compound method of buffer solution PBS used and PBSt is as follows:
Every liter of PBS is by NaCl 8.0g, KCl 0.2g, KH
2pO
40.2g, Na
2hPO
4.12H
2o 2.9g, add steaming water and be settled to 1000mL, and adjust pH to 7.4 is made.
Every liter of PBSt is by NaCl 8.0g, KCl 0.2g, KH
2pO
40.2g, Na
2hPO
4.12H
2o 2.9g, Tween-20 500uL, add steaming water and be settled to 1000mL, and adjust pH to 7.4 is made.
The immune affinity sorbent of embodiment 1, preparation enrichment ochratoxin A
The present embodiment has provided the conjugate that obtains by the ochratoxin A polyclonal antibody with containing the amino solid phase carrier coupling preparation method as the immune affinity sorbent of enrichment ochratoxin A.The method comprises:
One, the preparation of ochratoxin A specific polyclonal antibody
1, the preparation of ochratoxin A holoantigen and envelope antigen:
(1) preparation of ochratoxin A-NHS active ester (haptens)
1) get 50mg iodoacetic acid (0.269mmol) and 80mg N-hydroxy-succinamide (NHS) and (0.695mmol) be dissolved in 500uL oxolane (THF), obtain solution 1; By 120mg N, N-dicyclohexyl carbodiimide (DCC) (0.582mmol) is dissolved in 500uL THF, obtains solution 2.Solution 1 is mixed with solution 2, is that the shaking table that the 100rpm radius of turn is 200mm carries out condensation reaction 30min in room temperature (25 ℃) dark at rotating speed, and then the centrifugal 2min of 10000g, get supernatant and make NHS-iodacetyl active ester, standby.
2). by 50mg(0.124mmol) ochratoxin A thoroughly is dissolved in 500 μ LTHF and obtains ochratoxin A solution; the supernatant of step 1) (NHS-iodacetyl active ester) is joined in ochratoxin A solution; mix; be to carry out substitution reaction 1 hour on the 100rpm radius of turn shaking table that is 200mm at 20 ℃ of rotating speeds, obtain ochratoxin A-NHS active ester.
(2) preparation of ochratoxin A holoantigen and envelope antigen
Get 50mg carrier protein-keyhole limpet hemocyanin (keyhole limpet hemocyanin) (KLH) or bovine serum albumin(BSA) (BSA), be dissolved in the 5mL0.1mol/L sodium carbonate liquor, making pH is 8.3, add the ochratoxin A of step (1)-NHS active ester, mix, room temperature (20-25 ℃) is that the shaking table that the 100rpm radius of turn is 200mm carries out substitution reaction 1 hour in rotating speed.Product is crossed desalting column G25Sephadex(desalination step: with 10 times of volume distilled water washing desalting columns, add sample, after sample adds filler fully, add distilled water, discard empty amount volume (between filler particles and intragranular volume), the efflux of collection is exactly the sample after desalination, obtains the conjugate of ochratoxin A and carrier protein, as the ochratoxin A holoantigen, as the immunogene for preparing the ochratoxin A polyclonal antibody.The conjugate called after ochratoxin A-KLH of the ochratoxin A that is KLH by carrier protein-NHS active ester and carrier protein, the conjugate called after ochratoxin A-BSA of the ochratoxin A that is BSA by carrier protein-NHS active ester and carrier protein.Packing is preserved, for animal immune.
2, the preparation of ochratoxin A polyclonal antibody:
1) animal immune program:
The ochratoxin A prepared by step 1-BSA solution (solution that the ochratoxin A-BSA prepared with distilled water dissolving step 1 obtains) is as the vaccine immunity new zealand white rabbit, in vaccine, the concentration of ochratoxin A-BSA is 1mg/mL, every rabbit of first immunisation adds the 1mL Freund's complete adjuvant with the 0.5mL vaccine and mixes multi-point injection, every rabbit of booster immunization adds 0.75mL PBS with the 0.25mL vaccine and adds the 1mL Freunds incomplete adjuvant and mix, divide 2 injections, after first immunisation, at interval of 2 weeks booster immunizations once, the ELISA method for serum that gathers in 35-40 days (is diluted to 1ug/mL by the ochratoxin A-BSA of step 1, by the 100ul/ hole, be added on 96 orifice plates, 37 ℃ of standing coated 1h, take out, wash 3 times with PBSt, all pat dry at every turn.Add the 1%BSA100ul/ hole, 37 ℃ of standing sealing 1h, take out, and with PBSt, washes 3 times, all pats dry at every turn.Add PBS by the 100ul/ hole, by the PBS emulsion containing 0.1%BSA, press 1:500 dilution antiserum, add the dilute serum of 50ul1:500, mix, get 50ul and be added to the second hole ... the like, last hole does not add antibody, as blank, after antibody adds, 37 ℃ of standing reaction 1h, take out, wash 3 times with PBSt, all pat dry at every turn.By the 100ul/ hole, add two to resist, 37 ℃ of standing reaction 0.5h, take out, and with PBSt, washes 3 times, all pats dry at every turn.Add developer TMB by the 100ul/ hole, 37 ℃ of standing reaction 15min, take out, add the stop buffer cessation reaction by the 100ul/ hole, OD450 wavelength readings on ELIASA, dilution factor corresponding to the 1/2OD value of maximum OD450 value of usining is as antibody titer) detect titre, serum titer carries out the preparation of productivity serum after reaching 1:6400.
2) antiserum preparation
Gather antiserum from ear's vein, 20mL/ rabbit, serum is standing 1 hour in 4 ℃ after gathering.And then the centrifugal 10min of 5000g, collect supernatant, obtain antiserum (immune serum).
3) preparation of antigen affinity chromatograph filling:
By the ochratoxin A of step 1-NHS active ester under the pH8-9 condition directly with containing amino Ago-Gel (G Bios, 786-066) in the 50%(concentration expressed in percentage by volume) aqueous acetone solution in react, room temperature (20-25 ℃) is that shaking table that the 100rpm radius of turn is 200mm carries out substitution reaction and spends the night in rotating speed, generates ochratoxin A-agarose.Inferior daily PBS washes away filler impurity.Obtain ochratoxin A antigentic specificity affinity chromatograph filling, for separating of purification ochratoxin A polyclonal antibody.
4) polyclonal antibody is purified
By 500mL step 2) immune serum flow through the made affinity chromatograph filling post of step 3).After impurity is removed in the PBSt cleaning, the acetic acid aqueous solution wash-out that the anti-ochratoxin A antibody of the specificity on post is 3% by concentration expressed in percentage by volume.The antibody of wash-out removes acetic acid with the bag filter dialysis, and freeze-drying, obtain the ochratoxin A polyclonal antibody.
Two, the immune affinity sorbent of enrichment ochratoxin A preparation
By 50mL concentration, be that the 0.1mol/L sodium carbonate liquor dissolves the ochratoxin A polyclonal antibody that the 1g step 1 is purified, with 40mL containing amino Ago-Gel (G Bios, 786-066) the glass chromatography column room temperature downflow system circular response that is 90mL at volume 30 minutes.After adding the dissolving of 50mg sodium borohydride to mix, turn standing 30 minutes of 4 ℃ of refrigerators, after cleaning with 500mL PBSt, then clean with 100mL PBS, natural drip-dry, add equal-volume glycerine, mixes, and the immunity of enrichment ochratoxin A affine absorption-20 ℃ saves backup.
The affine absorption of immunity of getting the above-mentioned enrichment ochratoxin A of 40ml is packed into as filler in the affinity chromatography plastic column, filler two adds respectively the sieve plate that 1 aperture is 50 μ m, and sieve plate just contacts after (not pressing filler) immune affinity chromatographic column that obtains the affine absorption of immunity of 40ml enrichment ochratoxin A with filler.With PBS balance pillar, then add the ochratoxin A Anti-TNF-α liquid solution (containing 1000mg ochratoxin A polyclonal antibody) of purifying by the 50ml step 1 of PBS preparation, under natural gravity, flow out, make the pillar state that reaches capacity.Clean pillar with PBSt, unconjugated antibody is washed off.Application BCA method detected the content of the ochratoxin A polyclonal antibody before and after post, and the experiment triplicate, calculate the amount that is combined in the antibody on agarose.
Result shows that the ochratoxin A polyclonal antibody of unconjugated step 1 purifying is 150mg, the ochratoxin A polyclonal antibody of the step 1 purifying of actual coupling is 850mg, the antibody adsorbance of the immune affinity sorbent of this enrichment ochratoxin A (antibody density) average out to 21.25mg/mL.
The immune affinity chromatographic column of embodiment 2, enrichment ochratoxin A and Characteristics Detection thereof
One, the immune affinity chromatographic column of preparation enrichment ochratoxin A
Getting the immune affinity sorbent of the enrichment ochratoxin A of 25 μ L embodiment 1 preparations packs in the affinity chromatography plastic column that capacity is 200 μ L as filler, filler two adds respectively the sieve plate that 1 aperture is 50 μ m, obtains the immune affinity chromatographic column of the enrichment ochratoxin A of 25 μ L after compression.
The holding conditions of the immune affinity chromatographic column of this enrichment ochratoxin A: with the phosphate buffer that contains 0.01% Sodium azide and 0.1%BSA, preserve under 3-8 ℃, can not be freezing.The term of validity of this product is 12 months.
Two, the Characteristics Detection of the immune affinity chromatographic column of enrichment ochratoxin A
This step sample-loading buffer used is PBS.
This step rinsing liquid used is PBSt.
This step eluent used is acetic acid and methanol aqueous solution, and the solvent of described acetic acid and methanol aqueous solution is water, and solute is acetic acid and methyl alcohol, and the concentration expressed in percentage by volume of acetic acid is 3%, and the concentration expressed in percentage by volume of methyl alcohol is 80%.
1, the volume test of the immune affinity chromatographic column of enrichment ochratoxin A
Get the immune affinity chromatographic column measuring column capacity as follows of the enrichment ochratoxin A of the 25 μ L that prepare according to the condition of the step 1 storage step 1 of 12 months: affinity chromatographic column is taken out to equilibrium at room temperature 10min; With 5mLPBS balance pillar, then the ochratoxin A standard items (Sigma that the concentration that adds 5mL to prepare with PBS is 2ug/mL, O1877), under natural gravity, flow out, make the pillar state (criterion is: in efflux, ochratoxin A is identical with sample adding liquid concentration) that reaches capacity.Clean affinity chromatographic column with 10mL PBS again, remove interference impurity.Finally add 1mL eluent wash-out, collect eluent, detect according to GB/T25220-2010 by the HPLC method, calculate dynamic column capacity (referring to the obtained the maximum absorption of every milliliter of immunosorbent (or bed volume) to determinand).
The result of three repeated experiments shows the column capacity average out to 52 μ g/mL fillers of immune affinity chromatographic column of the enrichment ochratoxin A of this 25 μ L.The accumulation ability of immune affinity chromatographic column that this enrichment ochratoxin A is described is very strong, the ochratoxin A of the filling adsorption 52 μ g of average 1mL, and the accumulation rate of the immune affinity sorbent of enrichment ochratoxin A of the present invention is 52ug/mL.
2, the rate of recovery of the immune affinity chromatographic column of enrichment ochratoxin A test
Ochratoxin A standard items (sigma, O1877) are made respectively to ochratoxin A solution that concentration is 2ng/mL, 20ng/mL, 100ng/mL as testing sample with sample-loading buffer, and using sample-loading buffer as blank testing sample.According to the method for step 1, use the immune affinity chromatographic column of the enrichment ochratoxin A of the 25 μ L that prepare according to the condition of the step 1 storage step 1 of 12 months to detect the quality of the ochratoxin A in testing sample.
The result of twice repeated experiments shows: the ochratoxin A solution that 1. 50mL concentration is 2ng/mL is as testing sample, and the ochratoxin A eluted with eluent is respectively 80.5ng, 80.3ng, and average recovery rate is 80.4%;
2. the ochratoxin A solution that 5mL concentration is 20ng/mL is as testing sample, and the ochratoxin A eluted with eluent is respectively 90.2ng, 89.6ng; Average recovery rate is 89.9%;
3. the ochratoxin A solution that 1mL concentration is 100ng/mL is as testing sample, and the ochratoxin A eluted with eluent is respectively 83ng, 85ng, and average recovery rate is 84%;
4. blank testing sample eluent wash-out, do not detect ochratoxin A.
The immune affinity chromatographic column that the enrichment ochratoxin A is described has reached more than 80% the rate of recovery of ochratoxin A.
3, marginal test
Ochratoxin A standard items (sigma, O1877) are made respectively to ochratoxin A solution that concentration is 100ng/L, 1000ng/L as testing sample with sample-loading buffer, and using sample-loading buffer as blank testing sample.According to the method for step 1, use the immune affinity chromatographic column of the enrichment ochratoxin A of the 25 μ L that prepare according to the condition of the step 1 storage step 1 of 12 months to detect the quality of the ochratoxin A in testing sample.
The result of three repeated experiments is: the ochratoxin A solution that 1. 10mL concentration is 100ng/L is as testing sample, and the ochratoxin A eluted with eluent is respectively 1.3ng, 0.9ng, 1.0ng, average out to 1.07ng; 2. the ochratoxin A solution that 2mL concentration is 1000ng/L is as testing sample, and the ochratoxin A eluted with eluent is respectively 2.6ng, 2.1ng, 2.4, average out to 2.4ng; 3. blank testing sample eluent wash-out, do not detect ochratoxin A.The sensitivity that the immune affinity chromatographic column of this enrichment ochratoxin A is described can reach 100ng/L, and after enrichment is concentrated, sample reaches the requirement of detecting instrument.
The immune affinity chromatographic column of embodiment 3, enrichment ochratoxin A and colloidal gold test strip coupling are carried out qualitative test to testing sample
The immune affinity chromatographic column for preparing the enrichment ochratoxin A of 10 25 μ L, the preparation method of every is as follows: the immune affinity sorbent of getting the enrichment ochratoxin A of 25 μ L embodiment 1 preparations is packed in the affinity chromatography plastic column that capacity is 1mL as filler, filler two adds respectively the sieve plate that 1 aperture is 50 μ m, obtains the immune affinity chromatographic column of the enrichment ochratoxin A of 25 μ L after compression.
The immune affinity chromatographic column of 5 25 μ L enrichment ochratoxin As of random sampling, equilibrium at room temperature 10min; With 5mL PBS balance pillar, standby.By ochratoxin A standard items (sigma, O1877) make respectively ochratoxin A solution that concentration is 0.01ng/mL, 0.1ng/mL, 1.0ng/mL, 10.0ng/mL as testing sample with PBS, and using PBS as blank testing sample, carry out by the following method affinity chromatography.By the above-mentioned testing sample difference of 1mL upper prop, coutroi velocity is 0.05mL/s, collects efflux (filtered solution).Loading is complete, uses the PBS balance, and the acetic acid aqueous solution (with saturated tris-base solution, neutralizing pH=7.0-7.5) that the 100 μ L concentration expressed in percentage by volumes of then usining are 3% carries out wash-out as eluent and collects the liquid 100 μ L that elute.
With the ochratoxin A colloidal gold test strip of susceptibility 5ng/mL, (the ochratoxin A solution of above-mentioned 0.01ng/mL, 0.1ng/mL, 1.0ng/mL and 10.0ng/mL and PBS, the efflux of each testing sample, the liquid that each testing sample elutes AN-003) detect according to the specification of kit in Wuxi Andi Biological Engineering Co., Ltd.Test result shows, colloidal gold test strip is negative to ochratoxin A solution and the PBS testing result of 0.01ng/mL, 0.1ng/mL and 1.0ng/mL, testing result to the ochratoxin A solution of 10.0ng/mL is positive, illustrates that colloidal gold test strip conforms with product performance.The filtered solution that colloidal gold test strip is crossed after post all 5 kinds of samples all is negative.The immune affinity chromatographic column that this enrichment ochratoxin A is described can effectively be captured the ochratoxin A molecule.
The testing result that colloidal gold test strip carries out as sample the liquid that above-mentioned affinity chromatography elutes to sample-loading buffer PBS and 0.01ng/mL ochratoxin A solution is negative, the testing result that the ochratoxin A solution of 0.1ng/mL, 1.0ng/mL, 10.0ng/mL is carried out to the liquid that above-mentioned affinity chromatography elutes as sample is positive, the results are shown in Figure 1, illustrate: 1) antibody on the immune affinity chromatographic column of this enrichment ochratoxin A is stable, do not come off, do not cause the false positive come off because antibody is unstable.2) immune affinity chromatographic column of this enrichment ochratoxin A can play enrichment, the sample stoste of the 0.1ng/mL of 1mL negative (lower than the detection limit of colloidal gold test strip),, can be picked up colloidal gold test strip and be detected after 100 μ L through the immune affinity chromatographic column purification enrichment of this enrichment ochratoxin A.Explanation, after the immune affinity chromatographic column enrichment of enrichment ochratoxin A of the present invention, detects lower limit and can reach 0.1ng/mL.
The using method of the immune affinity chromatographic column of embodiment 4, enrichment ochratoxin A
Rinsing liquid in this embodiment is PBSt, and described eluent is acetic acid and methanol aqueous solution, and the solvent of described acetic acid and methanol aqueous solution is water, and solute is acetic acid and methyl alcohol, and the concentration expressed in percentage by volume of acetic acid is 3%, and the concentration expressed in percentage by volume of methyl alcohol is 80%.
A) pre-treatment of sample:
Cereal/Feed Sample: sample is ground and, by the testing sieve of 1mm, do not want pulverize.Take the sample of 5g (being accurate to 0.01g) grinding in the 15mL centrifuge tube, add 1g sodium chloride and 10mL extract (80% methanol aqueous solution), twist good lid, concussion at a high speed, extraction 5min; Then the centrifugal 5min of 4000rpm, accurately pipette the 4mL supernatant in the centrifuge tube of 50mL, adds PBS, and adjust pH to 7.4 is made and is diluted to scale, mixes, and through glass fiber filter paper, filters, and obtains amounting to 10mL for the sample solution of upper prop.
B). the immune affinity chromatographic column of the enrichment ochratoxin A of embodiment 2 preparations is moved on to room temperature (22-25 ℃) from 4 ℃ of refrigerators, after balance 10min, take out the top stopper, and pull out the below plug, with 2mL PBS washing 2 times.
C). the immune affinity chromatographic column of the enrichment ochratoxin A of processing through step b) with the eluent washing of 0.2mL, wash ochratoxin A antibody residual on filler off, after draining off, eluent is washed till neutrality with PBS.
D). the sample solution of step a) gained is crossed to post, and flow control is at 0.05mL/s.
E). after liquid drains fully, with rinsing liquid washing 2 times, each 2mL.
F). after liquid drains fully, carry out wash-out with eluent, the liquid eluted can be directly used in HPLC and detect analysis, perhaps the liquid eluted is dried up to rear using with nitrogen and redissolve liquid (10% methyl alcohol-0.5%BSA-PBSt) by the residue dissolving, this dissolved matter can be for ELISA testing cassete and colloidal gold strip test.Wherein, the composition of redissolution liquid is 10% for add methyl alcohol and BSA in PBSt solution to the methyl alcohol volume content, and BSA content is 0.5% liquid obtained.
The immune affinity chromatographic column of the enrichment ochratoxin A of embodiment 5, embodiment 2 detects the corn containing ochratoxin A
A) get not containing the corn kernel grinding of ochratoxin A and by the testing sieve of 1mm, not pulverize.Take the sample of 5g (being accurate to 0.01g) grinding in the 15mL centrifuge tube, add ochratoxin A standard items (sigma, O1877), the content that makes ochratoxin A in sample is 0.1ng/g, add 1g sodium chloride and 10mL extract (80% methanol aqueous solution), twist good lid, concussion at a high speed, extraction 5min; Then the centrifugal 5min of 4000rpm, accurately pipette the 4mL supernatant in the centrifuge tube of 50mL, adds PBS and be diluted to scale, mixes, and through glass fiber filter paper, filters, and obtains the sample solution for upper prop.
B). the immune affinity chromatographic column of the enrichment ochratoxin A of embodiment 2 preparations is moved on to room temperature (22-25 ℃) from 4 ℃ of refrigerators, after balance 10min, take out the top stopper, and pull out the below plug, with 2mL PBS washing 2 times.
C). the immune affinity chromatographic column of the enrichment ochratoxin A of processing through step b) with the eluent washing of 0.2mL, wash ochratoxin A antibody residual on filler off, after draining off, eluent is washed till neutrality with PBS.
D). the sample solution of step a) gained is crossed to post, and flow control is at 0.05mL/s.
E). after liquid drains fully, with rinsing liquid (with embodiment 4), wash 2 times each 2mL.
F). after liquid drains fully, with eluent (with embodiment 4), carry out wash-out, obtain the ochratoxin A solution of enrichment.
G). application GB detection method: in GB/T25220-2010 grain and oil detection grain, the mensuration high performance liquid chromatography of ochratoxin A and fluorimetry are measured the content of ochratoxin A in sample.
Claims (10)
1. the method for the immune affinity sorbent of preparation enrichment ochratoxin A, comprise the ochratoxin A polyclonal antibody and contain the step that amino solid phase carrier coupling obtains the immune affinity sorbent of enrichment ochratoxin A;
Described ochratoxin A polyclonal antibody is according to the preparation of the method that comprises the steps: the conjugate of ochratoxin A-NHS active ester and carrier protein of take obtains antiserum as the immunogen immune non-human mammal, and from described antiserum, purifying obtains described enrichment ochratoxin A polyclonal antibody.
2. method according to claim 1, it is characterized in that: described ochratoxin A-NHS active ester is that ochratoxin A passes through N, the active ester that N-dicyclohexyl carbodiimide and N-hydroxy-succinamide form.
3. method according to claim 2 is characterized in that: described ochratoxin A-NHS active ester is according to the method preparation comprised the steps:
1) by iodoacetic acid, N-hydroxy-succinamide and N, the N-dicyclohexyl carbodiimide carries out condensation reaction in the dark, obtains NHS-iodacetyl active ester;
2) described NHS-iodacetyl active ester and ochratoxin A are carried out to substitution reaction, obtain ochratoxin A-NHS active ester.
4. method according to claim 3, it is characterized in that: be described 1) by iodoacetic acid, N-hydroxy-succinamide and N, the N-dicyclohexyl carbodiimide is with the mol ratio of 269:695:582, in oxolane, 20 ℃-25 ℃ are carried out condensation reaction in the dark, after completion of the reaction, centrifugal with 10000g, get supernatant and obtain NHS-iodacetyl active ester;
Described 2) for the tetrahydrofuran solution of ochratoxin A and described supernatant are mixed, carry out substitution reaction at 20-25 ℃, obtain described ochratoxin A-NHS active ester.
5. according to arbitrary described method in claim 1-4, it is characterized in that: the method that purifying obtains described enrichment ochratoxin A polyclonal antibody from described antiserum comprises: using described ochratoxin A-NHS active ester and the described conjugate obtained containing amino solid phase carrier coupling as the affinity chromatography medium of the described ochratoxin A Anti-TNF-α of purifying, from described antiserum, by affinitive layer purification, obtain described ochratoxin A polyclonal antibody; And/or
The described ochratoxin A polyclonal antibody of the acetic acid aqueous solution wash-out that is 3% by concentration expressed in percentage by volume in described affinity chromatography.
6. according to arbitrary described method in claim 1-5, it is characterized in that: described is containing amino Ago-Gel or for containing amino sephadex containing amino solid phase carrier; And/or,
Described carrier protein is hemocyanin or bovine serum albumin(BSA).
7. the immune affinity sorbent of the enrichment ochratoxin A that in claim 1-6 prepared by arbitrary described method.
8. the immune affinity chromatographic column of the enrichment ochratoxin A that the immune affinity sorbent claimed in claim 7 of take is filler.
9. the kit that contains the enrichment ochratoxin A of immune affinity sorbent claimed in claim 7 or immune affinity chromatographic column claimed in claim 8.
10. immune affinity sorbent claimed in claim 7, immune affinity chromatographic column claimed in claim 8 or the application of kit claimed in claim 9 in the enrichment ochratoxin A.
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| CN105842454A (en) * | 2015-01-14 | 2016-08-10 | 北京康诺生物科技有限公司 | Oriented immunomagnetic beads of ochratoxin and preparation method and application of oriented immunomagnetic beads |
| CN110672852A (en) * | 2018-07-02 | 2020-01-10 | 中国医学科学院药用植物研究所 | Flow type magnetic ball detection method for ochratoxin A in traditional Chinese medicine |
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